High-performance liquid chromatography (HPLC) is widely used to separate organic mixtures. Ultraviolet, refractive index and fluorescence detectors most commonly used with HPLC are not as powerful as infrared (IR), mass spectroscopy (MS) and NMR for qualitative analysis of the HPLC eluents. While infrared and mass spectroscopy have been successfully coupled with gas chromatography (GC) to provide both separation and powerful spectroscopic analysis, efforts to similarly couple HPLC to one of the preferred spectroscopic techniques have not been as successful.
After intensive development HPLC coupled mass spectroscopy is commercially available. Efforts to couple HPLC on-line to infrared spectroscopy have been hampered by solvent interferences. NMR, which provides unequaled structural information and which has sample requirements more reasonably matched to HPLC, would appear to be the preferred spectroscopic method to couple with HPLC.
Efforts to couple these two analytical methods have been hampered primarily by the low sensitivity of the NMR detector. Since the initial demonstration of on-line coupled HPLC-NMR in 1978, some improvements in NMR sensitivity have been achieved through the use of high field superconducting solenoid NMR magnets rather than iron magnets. See David A. Laude and Charles L. Wilkins, "Nuclear Magnetic Resonance Detection for the On-line Identification of Liquid Chromatography Eluents," Trends in Analytical Chemistry, vol. 5, no. 9, 1986, 230-35. The other means by which an increase in NMR sensitivity has been sought is through the use of preparative or semi-preparative scale columns or analytical scale columns which have overloaded with excessive injection volume.
There are numerous disadvantages to these two alternative. The higher injection volumes associated with either of these methods can result in excessive band broadening Band broadening may be increased further if after the column a large dead space is required in order to meet the sample premagnetization requirement. Additional disadvantages with these two methods include a rapid degradation of column efficiency and the generation of excessive amounts of solvent which must be properly disposed of.
A better approach would provide improved NMR sensitivity while allowing for column injection limits in the low microgram region. The invention described herein provides a method for improving NMR sensitivity and lineshape while maintaining a low level of chromatographic band broadening and a high level of magnetic field homogeneity.